Polyethylene Glycol-Conjugated Glucocorticoid Prodrugs and Compositions and Methods Thereof

ABSTRACT

Polyethylene glycol (PEG)-conjugated glucocorticoid prodrugs, methods of preparation, and use for the treatment of diseases and disorders are disclosed. In particular, PEG-conjugated dexamethasone compounds and methods of using them for treating inflammatory and autoimmune diseases, including but not limited to lupus, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.17/188,309, filed on Mar. 1, 2021, now U.S. Pat. No. 11,583,541, whichis a Continuation of U.S. patent application Ser. No. 16/694,722, filedon Nov. 25, 2019, now U.S. Pat. No. 10,933,071, which is a Continuationof U.S. patent application Ser. No. 15/775,647, filed on May 11, 2018,now U.S. Pat. No. 10,485,809, which is the U.S. National Phase ofInternational Patent Application No. PCT/US2016/061728, filed on Nov.12, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/254,512, filed on Nov. 12, 2015,all of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to glucocorticoid prodrugs and methods ofusing the prodrugs for treating patients having an inflammatory disease,including but not limited to lupus.

BACKGROUND OF THE INVENTION

Lupus is a challenging autoimmune rheumatic disease in clinic for whichcurrent therapies are unsatisfactory with respect to both remissioninduction and unwanted toxicities. It is characterized by B and T cellhyperactivation, overproduction of autoantibodies, and the deposition ofimmune complexes in various tissues/organs. The symptoms of lupus arehighly heterogeneous including skin rash, arthritis, pericarditis,neuropsychiatric disorders and nephritis. It is estimated that 1.5million of Americans are affected by lupus and the number of patients iscontinuously increasing.

Lupus nephritis (LN), one of the most devastating complications oflupus, and the leading cause of morbidity and mortality in lupuspatients, affects between 30-60% of lupus patients in terms ofimmunosuppression and direct mortality. In the US, approximately 35%adult lupus patients have clinical evidence of nephritis at the time ofdiagnosis, and an additional 15-25% will develop nephritis within 10years of diagnosis. LN is initiated by immune complex deposition withinthe glomeruli and tubules of the kidney and subsequent activation of theimmune effector cells (such as macrophages and neutrophils) that leadsto damage to renal tissues. If not properly managed, lupus nephritis canrapidly progress to impaired renal function and eventually causing renalfailure.

While clinicians have utilized many classes of drugs to manage lupus,only a few have been approved by US FDA specifically for the disease.They include aspirin, belimumab (or Benlysta®, a human monoclonalantibody that inhibits B-cell activating factor), antimalarials (e.g.chloroquine) and glucocorticoids (GC, e.g. prednisone, dexamethasone).Among these treatment options, GC is one of the most potent and widelyused drugs for lupus. In American College of Rheumatology (ACR)'s newguidelines for clinical management of lupus nephritis, the recommendedtreatment regimen consists of a pulse GC treatment followed bylow/high-dose daily GC plus an immunosuppressive medication. Compared tothe previous guidelines, new immunosuppressants (e.g. mycophenolatemofetil) have been added as alternatives to cyclophosphamide. Noalternatives, however, have been recommended for GC. Comparing to thewide applications of GC in most lupus symptoms, the clinical benefits ofbelimumab in treatment of lupus nephritis has not been well established.NSAIDs, on the other hand, are contraindicated for lupus nephritis dueto their renal toxicities.

Due to their potent anti-inflammatory efficacy and the lack of analternative therapy, GC continues to be the mainstay of clinicalmanagement of lupus. Some lupus pathologies, such as arthritis and skinrash can be treated effectively with short-term GC. More severe lupuscomplications, such as progressive nephritis necessitates long-term GCtherapy, which is frequently associated with serious side effectsinvolving the endocrine, cardiovascular, hematopoietic andmusculoskeletal systems. These adverse events contribute significantlyto morbidity among lupus patients.

The actions of GC are thought to be mediated through two distinctpathways: transactivation and transrepression. It has been postulatedthat transrepression primarily mediates the anti-inflammatory effects ofGC whereas transactivation is responsible for the GC-associated sideeffects. Compounds that can preferentially activate the transrepressionrelative to the transactivation pathway have been developed.Nevertheless, these compounds do not exhibit strict pathway selectivityand still elicit GC-related side effects.

SUMMARY OF THE INVENTION

To address the various foregoing problems, the present applicationdiscloses glucocorticoid (GC) prodrugs and methods of using them fortreatment of lupus and LN. These include a polyethylene glycol(PEG)-based macromolecular prodrug (PEG-DiDex) of dexamethasone (Dex),which self-assembles into micelles.

In one aspect, the present invention provides a compound of formula (I)or (XII):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

-   -   n is an integer from 3 to 500;    -   m is an integer from 1 to 5;    -   w is an integer from 1 to 5;    -   GC is a moiety of a glucocorticoid drug molecule    -   A is absent, C₁-C₆ alkylene, or C₆-C₁₀ arylene;    -   B is absent, NR⁴, O, or C(O);    -   D is absent, NR⁴, O, C(O), or CR⁵R⁵;    -   E is absent, C₁-C₆ alkylene, or a linker comprising a branched        structure capable of connecting to two or more R² groups, said        linker optionally comprising one, two, or more heteroatoms        independently selected from the group consisting of O, S, and N;    -   G is absent, NR⁴, or O;    -   P is absent or C(O);    -   Q is absent, C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   T is absent, C₁-C₆ alkylene, C₆-C₁₀ arylene, or C(O);    -   X is absent, O, S, or NR⁴;    -   Y is absent, C(O), C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   Z is absent, NR⁴, O, C₁-C₆ alkylene, or a linker comprising a        branched structure capable of connecting to one or more        dexamethasone moieties, said linker optionally comprising one or        more heteroatoms independently selected from the group        consisting of O, S, and N;    -   R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl,        5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each        group except H optionally substituted by one to five        substituents independently selected from the group consisting of        C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, oxo (═O), —NR^(a)R^(b),        —NO₂, —CN, —OR³, and —SR³; or alternatively, R¹ is        —CH₂-A-B-D-E-(R²)_(m);    -   R³ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R⁴ at each occurrence is independently H or C₁-C₄ alkyl;    -   R⁵ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R^(a) and R^(b) are each independently H or C₁-C₄ alkyl; and    -   wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z        is absent, its two available adjacent groups are single-bonded        to each other directly.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound according to any one embodimentdescribed herein, or a pharmaceutically acceptable salt, solvate, orprodrug thereof, and a pharmaceutically acceptable carrier, adjuvant,diluent, or vehicle.

In another aspect, the present invention provides a method of treatingan autoimmune disease and/or inflammatory disorder, comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of a compound according to any embodiment disclosedhere, or a pharmaceutically acceptable salt, solvate, or prodrugthereof.

In another aspect, the present invention provides a method of treating adisease or disorder associated with lupus, comprising administering to asubject in need of treatment a therapeutically effective amount of thepharmaceutical composition of any embodiment disclosed herein.

In another aspect, the present invention provides use of a compoundaccording to any embodiment or any embodiment combinations disclosedhere, or a pharmaceutically acceptable salt, solvate, or prodrugthereof, in the manufacture of a medicament for treatment of a diseaseor disorder, especially those associated with lupus.

The PEG-based macromolecular prodrug of dexamethasone (Dex) overcomesvarious challenges, for example, their accompanying severe toxicities,through GC prodrug nanomedicine development, in order to more fullyrealize the therapeutic potential of GC in clinical management of lupusnephritis. Without intending to be bound by theory, the presentinvention is in part based on a hypothesis that the Extravasation of thenanomedicine through Leaky Vasculature at inflammation and subsequentInflammatory cell-mediated Sequestration (ELVIS) would dramaticallyalter the pharmacokinetics/biodistribution (PK/BD) profile of the parentdrug, favoring specific accumulation in the inflamed tissues/organs, andenhanced molecular weight associated with the non-immunogenicity of PEGchains would potentially improve drug retention in kidney and prolongthe blood circulation time. Specifically, the amphiphilic moleculeself-assembles into micelles. When tested in lupus prone NZB/W F1 micewith severe nephritis, the monthly treatment demonstrated superiortherapeutic efficacy in improving kidney function than dose equivalentdaily Dex treatment, with no GC-associated side effect observed.

Other aspects, benefits, and advantages of the present invention will bebetter appreciated in view of the following drawings, detaileddescription, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the design of polyethylene glycol (PEG)-basedamphiphilic dexamethasone prodrug PEG-DiDex, which can self-assembleinto micelles.

FIG. 2 illustrates transmission electron microscope image of PEG-DiDexmicelles deposited on formvar/silicone monoxide coated 200 mesh coppergrids surface. The estimated average diameter of the micelles is ˜30 nm.

FIG. 3 illustrates the release of Dex from PEG-DiDex in acetate buffer(pH=5.0) at 37° C. Pluoronic F127 was added to create the sinkcondition.

FIGS. 4A and 4B illustrate monthly PEG-DiDex treatment results, whichdemonstrate superior therapeutic effect when compared to dose equivalentdaily Dex treatment of 28 wks old NZB/W F1 female mice with severenephritis. (A) Monthly PEG-DiDex treatment normalized albuminuria among60% of NZB/W F1 mice, while dose equivalent daily Dex treatment onlynormalized 18% at the end of 2-months treatment. PT=pretreatment. (B)Kaplan-Meier survival curves for PEG-DiDex, Dex and saline treatmentgroups are shown. Only PEG-DiDex treatment results in 100% survivalafter 2-month treatment.

FIGS. 5A, 5B, 5C, 5D, 5E, and 5F illustrate histological evaluation ofkidneys isolated at the 2-month treatment end points. The tissues wereformalin-fixed, sectioned (3 μm) and periodic-acid schiff (PAS) stainedfor visual examination and 4 point grading by a pathologist, who isblind to the group design. (A) PAS-stained kidney section from salinegroup. Bar=20 μm; (B) PAS-stained kidney section from Dex group. Bar=20μm; (C) PAS-stained kidney section from PEG-DiDex group. Bar=20 μm; (D)PAS-stained kidney section from NZW control group. Bar=20 μm; (E) Thefraction of mice in each group with mild, moderate and severe renaldisease is shown; (F) The percentage of abnormal glomeruli found in eachgroup is shown.

FIG. 6 illustrates treatment with PEG-DiDex, which attenuates seruminflammatory cytokines.

FIGS. 7A, 7B, 7C, 7D, 7E, and 7F illustrate that two months of PEG-DiDexmicelle treatment of NZB/W F1 mice did not lead to typicalglucocorticoid toxicities. (A) PEG-DiDex micelles treatment issignificantly better in preserving bone mineral density (BMD) than Dextreatment; (B) PEG-DiDex micelles treated mice have a trend of higherbone volume/tissue volume (BV/TV) than those from the Dex and salinegroups; (C) PEG-DiDex micelles treated mice have a significantly highertrabecular thickness (Tb.Th.) value than Dex and saline groups; (D)PEG-DiDex micelles treated mice have a significantly higher white bloodcell (WBC) count than Dex treated mice; (E) PEG-DiDex treatment did notcause significant reduction of total serum IgG level, while Dextreatment did, which is a sign of potential immune suppression; (F)Different from Dex treatment, PEG-DiDex treatment does not induceadrenal gland atrophy. The asterisk (*) indicates a statisticallysignificant difference (P<0.05).

FIG. 8 illustrates passive targeting of IRDye labeled PEG-DiDex tonephritis in NZB/W F1 mice. NZW mice were used as control. At differenttime points post tail vein injection (1 and 4 days), heart (he), lung(lu), kidney (kd), liver (lv), spleen (sp) and adrenal gland (ad) wereisolated after saline perfusion and subjected to near infrared imaging.Pseudo color-coded signal intensity reflect the level of PEG-DiDexwithin the organ examined.

FIG. 9 illustrates the impact of different treatment regiments on theserum anti-dsDNA IgG level. While Dex daily treatment significantlyreduced the antibody level, dose equivalent monthly PEG-DiDex treatmenthad not impact.

FIGS. 10A and 10B illustrate that PEG-DiDex ameliorates albuminuria,extends lifespan and attenuates development of severe nephritis. (A),Albuminuria data for mice in saline (n=12), Dex (n=11), and PEG-DiDex(n=11) treatment groups is illustrated at the pretreatment (PT) and8-week time points. The incidence of albuminuria at the 8-week timepoint for each group is shown (in %) in upper right corner of eachsub-section. (B), A Kaplan-Meier survival curve for each treatment groupis shown.

DETAILED DESCRIPTION OF THE INVENTION

The glucocorticoid prodrugs conjugated to polyethylene glycol have beenfound to possess superior therapeutic efficacy and greatly reducedtoxicity as compared with the parent drug.

Specifically, optical imaging, immunohistochemistry and flow cytometrystudies reveal that the near-infrared dye labeled PEG-DiDex micelleprimarily distributes to the inflamed kidneys after systemicadministration, with intraglomerular mesangial cells and proximal tubuleepithelial cells chiefly responsible for the intracellular sequestrationof the prodrug inside the kidneys. For efficacy and safety evaluation,the prodrug micelle was given monthly to NZB/W F1 female mice (28 weeksold) via tail vein injection. Dose equivalent daily dexamethasonephosphate sodium i.v. administration and saline were used as controls.When compared to Dex treatment, PEG-DiDex markedly improved the survivalof NZB/W F1 mice and is significant more effective in normalizingalbuminuria; no significant systemic toxicity of GCs (i.e. WBCreduction, total IgG reduction, adrenal gland atrophy and osteopenia)was observed in the prodrug treated group. PEG-DiDex treated animalsalso exhibit lower serum levels of proinflammatory cytokines (e.g.MCP-1, IFN-β, IFN-γ, etc.) and clear histological indication ofnephritis resolution after 2 months of treatment. But it has no impacton the serum anti-dsDNA antibody level. Collectively, these evidencessuggest that the novel prodrug micelle design of PEG-DiDex candramatically alter the biodistribution profile of Dex by passivelytargeting the drug to the inflamed kidneys. The nephrotropicdistribution pattern of PEG-DiDex potentiates and prolongs the localanti-inflammatory effect of Dex within kidney pathology. Its outstandingsafety profile may be attributed to the substantially reduced systemicexposure to Dex.

In one aspect, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

-   -   n is an integer from 3 to 500;    -   m is an integer from 1 to 5;    -   w is an integer from 1 to 5;    -   A is absent, C₁-C₆ alkylene, or C₆-C₁₀ arylene;    -   B is absent, NR⁴, O, or C(O);    -   D is absent, NR⁴, O, C(O), or CR⁵R⁵;    -   E is absent, C₁-C₆ alkylene, or a linker comprising a branched        structure capable of connecting to two or more R² groups, said        linker optionally comprising one, two, or more heteroatoms        independently selected from the group consisting of O, S, and N;    -   G is absent, NR⁴, or O;    -   P is absent or C(O);    -   Q is absent, C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   T is absent, C₁-C₆ alkylene, C₆-C₁₀ arylene, or C(O);    -   X is absent, O, S, or NR⁴;    -   Y is absent, C(O), C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   Z is absent, NR⁴, O, C₁-C₆ alkylene, or a linker comprising a        branched structure capable of connecting to one or more        dexamethasone moieties, said linker optionally comprising one or        more heteroatoms independently selected from the group        consisting of O, S, and N;    -   R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl,        5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each        group except H optionally substituted by one to five        substituents independently selected from the group consisting of        C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, oxo (═O), —NR^(a)R^(b),        —NO₂, —CN, —OR³, and —SR³; or alternatively, R¹ is        —CH₂-A-B-D-E-(R²)_(m);    -   R³ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R⁴ at each occurrence is independently H or C₁-C₄ alkyl;    -   R⁵ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R^(a) and R^(b) are each independently H or C₁-C₄ alkyl; and    -   wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z        is absent, its two available adjacent groups are single-bonded        to each other directly.

In some embodiments of this aspect, E is a linker comprising a branchedstructure capable of covalently bonding to two or more R² groups, saidlinker optionally comprising one or more heteroatoms independentlyselected from the group consisting of O, S, and N.

In some embodiments of this aspect, Z is a linker comprising a branchedstructure capable of covalently bonding to two or more dexamethasonemoieties, said linker optionally comprising one or more heteroatomsindependently selected from the group consisting of O, S, and N.

In some embodiments of this aspect, E and Z are each independentlyselected from the group consisting of absent, C1-C4 alkylene, an aminoacid-based linker, a citric acid-based linker, a glycerol-based linker,a tris(2-aminoethyl)amine-based linker, a pentaerythritol-based linker,and a pentetic acid-based linker, respectively having a formula asfollows:

wherein i and j are each independently 0 or an integer select from 1 to5.

In some embodiments of this aspect,

-   -   w is 1;    -   Z is absent or C₁-C₄ alkylene;    -   E is selected from the group consisting of:

In some embodiments of this aspect, m is 1; w is 1; and A, B, D, E, andZ are all absent, characterized by formula (II):

In some embodiments of this aspect, R¹ is CH₃; Y is C(O); X is NH; andT, Q, P, and G are all absent, characterized by formula:

In some embodiments of this aspect, A is C(O); B and D are absent; E isthe amino acid based linker; m is 2; w is 1; Z and Y are absent, and Xis NH, characterized by formula (III):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

-   -   i=0 or an integer from 1 to 5;    -   j=0 or an integer from 1 to 5;    -   G=absent, NR⁴, or O;    -   P=absent or C(O);    -   Q=absent, C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   T=absent, C₁-C₆ alkylene, C₆-C₁₀ arylene, or C(O);    -   X=absent, O, S, or NR⁴;    -   Y=absent or C₁-C₆ alkylene;    -   R¹═H or C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl,        or 5-10 membered heteroaryl, 5-10 membered heterocyclyl, each        group except H optionally substituted by one to three        substituents independently selected from the group consisting of        C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, oxo (═O), —NR^(a)R^(b),        —NO₂, —CN, —OR³, and —SR³;    -   R³ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R⁴ at each occurrence is independently H or C₁-C₄ alkyl;    -   R^(a) and R^(b) are each independently H or C₁-C₄ alkyl; and    -   wherein when any of groups G, P, Q, T, X, and Y is absent, its        two available adjacent groups are single-bonded to each other        directly.

In some embodiments of this aspect, i is 0; j is 2; E is a glutamic acidbased linker having a formula:

In some embodiments of this aspect, i is 0, j is 2, X is NH; and Q ismethylene, P is C(O), G is NH, the compound characterized by formula:

In some embodiments of this aspect, A is methylene, B is NH, m is 1, wis 2, E is a citric acid based linker, Z is NH, the compoundcharacterized by formula (IV):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

-   -   i=0 or an integer from 1 to 5;    -   j=0 or an integer from 1 to 5;    -   G=absent, NR⁴, or O;    -   P=absent or C(O);    -   Q=absent, C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   T=absent, C₁-C₆ alkylene, C₆-C₁₀ arylene, or C(O);    -   X=absent, O, S, or NR⁴;    -   Y=absent or C₁-C₆ alkylene;    -   R¹═H or C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl,        or 5-10 membered heteroaryl, 5-10 membered heterocyclyl, each        group except H optionally substituted by one to three        substituents independently selected from the group consisting of        C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, oxo (═O), —NR^(a)R^(b),        —NO₂, —CN, —OR³, and —SR³;    -   R³ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R⁴ at each occurrence is independently H or C₁-C₄ alkyl;    -   R^(a) and R^(b) are each independently H or C₁-C₄ alkyl; and    -   wherein when any of groups G, P, Q, T, X, and Y is absent, its        two available adjacent groups are single-bonded to each other        directly.

In some embodiments of this aspect, G, P, Q, T, X, and Y are all absent,the compound characterized by formula:

In some embodiments of this aspect, m is 3, w is 1, A is methylene, B isNH, D is C(O) and E is a pentaerythritol-based linker, the compound ischaracterized by formula (V):

In some embodiments of this aspect, Y is C(O); X is NH; and G, P, Q, andT are all absent, the compound characterized by formula:

In some embodiments of this aspect, the compound has a structure of anyone of formulas (VI)-(XI):

wherein R is selected from the group consisting of:

wherein k at each occurrence is independent an integer selected from 1to 10. In some embodiments, k is an integer selected from 1 to 8; insome preferred embodiments, k is an integer selected from 1 to 6; and insome more preferred embodiments, k is an integer selected from 1 to 4;and in some more preferred embodiments, k is an integer selected from 1to 2.

In some embodiments of this aspect, E is a glutamic acid based linkerhaving a formula:

In another aspect, the present invention provides a compound of formula(XII):

or a pharmaceutically acceptable salt, solvate, or prodrug thereof,wherein:

-   -   n is an integer from 3 to 500;    -   m is an integer from 1 to 5;    -   w is an integer from 1 to 5;    -   GC is a moiety of a glucocorticoid drug molecule;    -   A is absent, C₁-C₆ alkylene, or C₆-C₁₀ arylene;    -   B is absent, NR⁴, O, or C(O);    -   D is absent, NR⁴, O, C(O), or CR⁵R⁵;    -   E is absent, C₁-C₆ alkylene, or a linker comprising a branched        structure capable of connecting to two or more R² groups, said        linker optionally comprising one, two, or more heteroatoms        independently selected from the group consisting of O, S, and N;    -   G is absent, NR⁴, or O;    -   P is absent or C(O);    -   Q is absent, C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   T is absent, C₁-C₆ alkylene, C₆-C₁₀ arylene, or C(O);    -   X is absent, O, S, or NR⁴;    -   Y is absent, C(O), C₆-C₁₀ arylene, or C₁-C₆ alkylene;    -   Z is absent, NR⁴, O, C₁-C₆ alkylene, or a linker comprising a        branched structure capable of connecting to one or more        dexamethasone moieties, said linker optionally comprising one or        more heteroatoms independently selected from the group        consisting of O, S, and N;    -   R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl,        5-10 membered heteroaryl, or 5-10 membered heterocyclyl, each        group except H optionally substituted by one to five        substituents independently selected from the group consisting of        C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, oxo (═O), —NR^(a)R^(b),        —NO₂, —CN, —OR³, and —SR³; or alternatively, R¹ is        —CH₂-A-B-D-E-(R²)_(m);    -   R³ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R⁴ at each occurrence is independently H or C₁-C₄ alkyl;    -   R⁵ at each occurrence is independently H, C₁-C₄ alkyl, or C₁-C₄        haloalkyl;    -   R^(a) and R^(b) are each independently H or C₁-C₄ alkyl; and    -   wherein when any of groups A, B, D, E, G, P, Q, T, X, Y, and Z        is absent, its two available adjacent groups are single-bonded        to each other directly.

In this aspect, the term “moiety of a glucocorticoid drug molecule”refers to the drug portion of the prodrug linked with PEG throughvarious linkers through a C═N double bond as disclosed herein. Inparticular, these moieties of glucocorticoid drug molecules include, butare not limited to the following:

or the like.

In some embodiments of this aspect, the compound a structure selectedfrom formulae (XIII) to (XVIII):

wherein R comprises said moiety of a glucocorticoid drug molecule.

In some embodiments, the glucocorticoid drug molecule is selected fromthe group consisting of:

The size of PEG suitable for compounds of the present invention can varyin a range so that the prodrug can serve the purpose disclosed herein.Typical size can be in the range of 100 to 20,000 Da. molecular weight,or n can be in the range of about 3 to about 500. In some embodiments, nis in the range of 10 to 300. In some embodiments of this aspect, n is20 to 200. In some embodiments of this aspect, n is 10 to 100.

In some preferred embodiments of this aspect, n is 40-45; and in someparticular embodiments, n is 42.

As a person of ordinary skill in the art would understand, in anyaspects or embodiments of the compounds disclosed herein, any twoadjacent atoms or bonds must comply with the basic bond principles.While basic bond principles are compliant with, any potentialcombination of the limitations defined in any two or more embodimentsdisclosed herein are encompassed by the present invention.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound according to any one embodimentdescribed herein, or a pharmaceutically acceptable salt, solvate, orprodrug thereof, and a pharmaceutically acceptable carrier, adjuvant,diluent, or vehicle.

In some embodiments of this aspect, the pharmaceutical composition is inoral, nasal, ophthalmic drop, topical, buccal, sublingual, rectal,vaginal, intravenous, or other parenteral form.

In some embodiments of this aspect, the pharmaceutical composition is invaporization-ready, nebulization-ready, nanoparticle formulation, orliposomal formulation.

In some embodiments of this aspect, the composition of further comprisesa second therapeutic agent, including but not limited, a NSAID (e.g.Aspirin, Naproxen, Celebrex), a Glucocorticoid (e.g. Dexamethasone,Prednisone, Betamethasone), a DMARD (e.g. Methotrexate, Leflunomide,Sulfasalazine, Hydroxychoroquine), or the like.

In another aspect, the present invention provides a method of treatingan autoimmune disease and/or inflammatory disorder, comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of a compound according to any embodiment disclosedhere, or a pharmaceutically acceptable salt, solvate, or prodrugthereof.

In some embodiments of this aspect, the disease or disorder is systemiclupus erythematosus, lupus nephritis, minimal change disease, focalsegmental glomerulosclerosis, IgA nephropathy, transplant rejection,rheumatoid arthritis, osteoarthritis, psoriasis, ankylosing spondylitis,vasculitis, multiple sclerosis, systemic sclerosis, gout, uveitis,asthma, cystic fibrosis, chronic obstructive pulmonary disease, atopicdermatitis (eczema), sepsis, inflammatory bowel disease, trauma braininjury, spinal cord injury, ischemia reperfusion injury, heterotopicossification, or granuloma, etc.

In some embodiments of this aspect, the method of treating disease ordisorder further includes administering to the subject a secondtherapeutic agent, such as a NSAID (e.g. Aspirin, Naproxen, Celebrex), aGlucocorticoid (e.g. Dexamethasone, Prednisone, Betamethasone), a DMARD(e.g. Methotrexate, Leflunomide, Sulfasalazine, Hydroxychoroquine), abiologic (e.g. Belimumab, Etanercept, Anakinra, Infliximab, Rituximab,etc.), or the like.

In another aspect, the present invention provides a method of treating adisease or disorder associated with lupus, comprising administering to asubject in need of treatment a therapeutically effective amount of thepharmaceutical composition of any embodiment disclosed herein.

In some embodiments of this aspect, the subject treated is a mammaliananimal, such as human, dog, cat, horse, and so on. In a preferredembodiment, the subject is a human.

In another aspect, the present invention provides use of a compoundaccording to any embodiment or any embodiment combinations disclosedhere, or a pharmaceutically acceptable salt, solvate, or prodrugthereof, in the manufacture of a medicament for treatment of a diseaseor disorder, especially those associated with lupus.

The disease or disorder that can be treated using the present inventionincludes, but is not limited to, systemic lupus erythematosus, cutaneouslupus erythematosus, photosensitivity, cutaneous vascular disease,nonscarring alopecia, oral ulcer, nail and capillary changes,populonodular mucinosis, bullous lupus erythematosus, sweet syndrome,pyoderma gangrenosum, palisaded neutrophilic granulomatous dermatitis,aseptic meningitis, cerebrovascular disease, demyelinating syndrome,headache, movement disorder, myelopathy, seizure disorder, acuteconfusional state, anxiety disorder, cognitive dysfunction, mooddisorder, psychosis, Guilain-Barré syndrome, autonomic neuropathy,mononeuropathy, myasthenia gravis, cranial neuropathy, plexopathy,polyneuropathy, lupus nephritis, pericarditis, coronary vasculitis,coronary atherosclerosis, vasculitis, pleurisy, pleural effusion, acutelupus pneumonitis, diffuse alveolar hemorrhage, chronic interstitiallung disease shrinking lung syndrome, pulmonary hypertension,thromboembolism, cricoarytenoid arthritis, small airways disease,chronic active and lupoid hepatitis, Sjögren's syndrome, esophagitis,watermelon stomach, eosinophilic gastroenteritis, abdominal pain,intestinal thrombosis, inflammatory bowel disease, protein-losingenteropathy, fat malabsorption, Celiac Sprune, Chronic intestinalpseudo-obstruction, amyloidosis, peritoneal inflammation, pancreatitis,splenomegaly, autoimmune hemolytic anemia, immune thrombocytopenicpurpura, leukopenia, arthralgia, arthritis, tendon rupture, myositis,osteonecrosis, osteoporosis, etc.

Without being limited, in some embodiments, the molecular weight of PEGused in the present invention is in the range from about 200 to about10,000 Da. The glucocorticoid (GC) drug molecule can be conjugated to achain terminus, or both chain termini of PEG. In addition to linear PEG,branched PEG, brush like PEG or even dendritic PEG can also be used asthe prodrug carrier for conjugation with molecules such as Dex. Thenumber of drug molecule such as dexamethasone (Dex) can range from 1 to10 in each molecule of PEG prodrug conjugate. To increase the micellestability, the hydrophobic terminus may be crosslinked with weakchemical bonds.

In addition to dexamethasone, other glucocorticoids (e.g. prednisone,prednisolone, methylprednisolone, betamethasone, triamcinolone,beclometasone), anti-inflammatory agents, and low molecular weightimmune suppressant can be used to conjugate to PEG according to theprinciple and methods disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as are commonly understood by one of ordinaryskill in the art to which this invention pertains.

Unless otherwise specified, a reference to a particular compoundincludes all of its isomeric forms. For example, when a compound has aC═N—X moiety (X═NHR, OR, or R, wherein R is any suitable group, e.g., H,alkyl, aryl, or acyl, etc.), such as hydrazone (X═NHR), imine (X═R), oroxime (X═OR) derivatives, the X group can exist in syn- (E-) or anti-(Z-) configuration relative to the other portion of the molecule, inparticular the groups on both sides of the C═N bond, as would beunderstood by a person or ordinary skill in the art. When a compound hasmultiple such C═N—X moieties, multiple isomers would be possible. In thepresent disclosure, all such isomers, in pure or mixture forms or anycombinations thereof, are encompassed by the name or structure of suchcompounds, as would be understood by those of ordinary skill in thepertinent art, even though they are not explicitly called by names orshown in the structures presented. Unless otherwise specified, areference to a particular compound also includes ionic, salt, solvate(e.g., hydrate), protected forms, and other stereoisomers thereof, forexample, as discussed herein.

In the compounds disclosed herein, the atoms may exhibit their naturalisotopic abundances, or one or more of the atoms may be artificiallyenriched in a particular isotope having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberpredominantly found in nature. For example, substitution with heavierisotopes, such as replacing hydrogen (H) with deuterium (i.e., ²H or D)may afford certain therapeutic advantages resulting from greatermetabolic stability (e.g., increased in vivo half-life or reduced dosagerequirements) and hence may be preferred in some circumstances. Inaddition, certain isotopically-labeled compounds (e.g., with ³H and ¹⁴C)are useful in compound and/or substrate tissue distribution assays.Isotopically labeled compounds can generally be prepared by followingprocedures analogous to those disclosed in the Schemes and/or in theExamples below, by substituting an appropriate isotopically labeledreagent for a non-isotopically labeled reagent. The present invention ismeant to include all suitable isotopic variations of the compoundsdisclosed.

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

When the term “about” is applied to a number or parameter, the number orparameter can vary by ±10%, inclusive. As would be understood by aperson skilled in the art, when a parameter is not critical, a number isoften given only for illustration purpose, instead of being limiting.

The term “a,” “an,” or “the,” as used herein, represents both singularand plural forms. In general, when either a singular or a plural form ofa noun is used, it denotes both singular and plural forms of the noun.

The term “treating,” “treatment,” or “therapy,” or the like, as usedherein in the context of treating a disease or condition, pertainsgenerally to treatment and therapy of an animal subject, preferably ahuman, in which some desired therapeutic effect is achieved. Forexample, therapy can include the inhibition of the progress of thecondition, reduction in the rate of progress, a halt in the rate ofprogress, amelioration of the condition, absolute or partial preventionof a delayed complication, and cure of the condition. Treatment alsoincludes prophylactic measure as well as adjunct treatments to astandard treatment regimen established in the art.

The term “therapeutically effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosageform comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio.

It may be convenient or desirable to prepare, purify, and/or handle theactive compound in the form of a prodrug. The term “prodrug” as usedherein, pertains to a compound which, when metabolized, yields thedesired active compound or in itself is the active compound. Typically,the prodrug is inactive, or less active than the active compound, butmay provide advantageous handling, administration, or metabolicproperties. For example, some prodrugs are ethers or esters of theactive compound; during metabolism the ether group is cleaved to yieldthe active drug. Also, some prodrugs are activated enzymatically toyield the active compound, or a compound which, upon further chemicalreaction, yields the active compound. Thus, in the methods of treatmentof the present invention disclosed herein, the term “administering”shall encompass the treatment of the various conditions described withthe compound specifically disclosed or with a compound which may not bespecifically disclosed, but which converts to the specified compound invivo after administration to the patient. Metabolites of these compoundsinclude active species produced upon introduction of compounds of thisinvention into the mammalian subject.

Any of the compounds of the present invention may be contemplated foradministration to the human subject in the form of a drug, prodrug oreven active metabolite.

In particular, although the compounds disclosed herein are prodrugsthemselves relative to the parent drug compound, e.g., glucocorticoids(e.g. dexamethasone, prednisone, etc.), these compounds themselves mayexist in their prodrug forms. For example, any of the hydroxyl or aminogroups on the molecule may be protected by an acyl (e.g., acetyl) orother group that can be readily hydrolyzed under the physiologicalconditions to become the parent polyethylene glycol-glucocorticoid(PEG-GC) drug compound. Such additional level of prodrug may even bepreferred under certain circumstances to further control the releasemode and rate of the parent drug compound in a subject.

As noted herein, the salts of the compounds of this invention refer tonon-toxic “pharmaceutically acceptable salts.” Other salts may, however,be useful in the preparation of the compounds according to the inventionor of their pharmaceutically acceptable salts. When the compounds of thepresent invention contain a basic group, salts encompassed within theterm “pharmaceutically acceptable salts” refer to non-toxic salts whichare generally prepared by reacting the free base with a suitable organicor inorganic acid. Representative salts include any such salt known inthe art. Where compounds of the present invention carry an acidicmoiety, suitable pharmaceutically acceptable salts thereof may includealkali metal salts, e.g., sodium or potassium salts; alkaline earthmetal salts, e.g., calcium or magnesium salts; and salts formed withsuitable organic ligands, e.g., quaternary ammonium salts.

To treat a human patient, an effective amount of one or more compoundsof the present invention, or a pharmaceutically-acceptable salt thereof,is administered to the human subject in need so as to promote exposureto or contact of the tissue at risk or the targeted region of the bodyor nerves, synapses, or neuromuscular junctions, or organ systemsincluding but not limited to the autonomic and central nervous systems.Effective dosage forms, modes of administration and dosage amounts maybe determined empirically, and making such determinations is within theskill of the art.

As discussed herein, the PEG-GC drug compounds disclosed herein can beadministered in such oral dosage forms as tablets, capsules (each ofwhich includes sustained release or timed release formulations), pills,powders (e.g., reconstitutable lyophilized powder), micronizedcompositions, granules, elixirs, tinctures, suspensions, ointments,vapors, liposomal particles, nanoparticles, syrups and emulsions.Likewise, they may also be administered in intravenous (bolus orinfusion), intraperitoneal, topical (e.g., dermal, epidermal,transdermal, ophthalmically such as ocular eyedrop), intranasally,subcutaneous, inhalation, intramuscular or transdermal (e.g., patch,microneedles) form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. Again, the ordinarily skilledphysician, veterinarian or clinician or a clinical pharmacist mayreadily determine and prescribe the effective amount of the drugrequired to prevent, counter or arrest the progress of the condition.

As noted herein, the compounds of the present invention may be used incombination with other drugs or therapies having similar orcomplementary effects to those of the compounds disclosed herein. Theindividual components of such combinations can be administeredseparately at different times during the course of therapy orconcurrently in divided or single combination forms to patients orregions of such patients in need of such therapy. The instant inventionis therefore to be understood as embracing all such regimes ofsimultaneous or alternating treatment and the term “administering” is tobe interpreted accordingly.

As used herein, the term “composition,” “pharmaceutical composition,” orthe like, is intended to encompass a product comprising the specifiedingredients in the specified amounts, as well as any product whichresults, directly or indirectly, from combination of the specifiedingredients in the specified amounts.

The amount of the active ingredient(s) which will be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration andall of the other factors described above. The amount of the activeingredient(s) which will be combined with a carrier material to producea single dosage form will generally be that amount of the activeingredient(s) which is the lowest dose effective to produce atherapeutic effect.

Pharmaceutical formulations of the present invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. Formulations of theinvention suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders (e.g.,reconstitutable lyophilized powder), granules, or as a solution or asuspension in an aqueous or nonaqueous liquid, or as an oil-in-water orwater-in-oil liquid emulsion, or as an elixir or syrup or tincture, oras pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and the like, each containing a predetermined amountof the active ingredient(s). The active ingredient(s) may also beadministered as a bolus, electuary or paste.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampoules and vials, or in specialized capsulesfor vapor or nebulized administration and may be stored in a lyophilizedcondition requiring only the addition of the sterile liquid carrier, forexample water or oil for injection, immediately prior to use.Extemporaneous injection solutions and suspensions maybe prepared fromsterile powders, granules and tablets of the type described above.

PEG-DiDex is an amphiphilic macromolecular prodrug with a Dex dimerconstitutes the hydrophobic section. A mPEG 1900 was employed as thehydrophilic section. The utility of this short PEG as the carrier willnot only reduce WBC internalization but also significantly reduce theserum half-life of the prodrug, which may greatly limit the prodrug'sdistribution to the mononuclear phagocyte system (MPS). Due to the useof PEG as the drug carrier, PEG-DiDex activation is slow (FIG. 3 ),which may be helpful in maintaining a low serum concentration of freeDex. The amphiphilic design would allow the prodrug to self-assembleinto micelles (FIG. 1 and FIG. 2 ) so that it would not be clearedthrough kidney too fast and overwhelm the renal cell sequestrationcapacity. The synthesis of PEG-DiDex is straightforward with high yieldat each step. Due to the use of hydrazone linker as the prodrugactivation trigger, PEG-DiDex will have multiple syn/anti-hydrazoneisomers, which makes the interpretation of the NMR spectrum difficult.To minimize formation of multiple isomers, in some embodiments symmetricbranching structures such as citric acid may be used instead of glutamicacid. Furthermore, to minimize formation of prodrug with polydispersemolecular weight due to the use of conventional PEG, in someembodiments, it may be preferable to use single molecular weightdiscrete PEG (dPEG®), which has become commercially available. Differentfrom traditional polymeric prodrug design, the use of dPEG® in thesynthesis of PEG-DiDex will yield a product with a single molecularweight, which will remove potential regulatory hurdles during theproduct development process.

Treatment of NZB/W F1 mice with severe nephritis with PEG-DiDex monthlyeffectively attenuated albuminuria and maintained 100% animal survivalfor the entire experiment duration. On the other hand, dose equivalentdaily Dex treatment only presented with moderate efficacy and 80%survival (FIG. 4 ). These observations were further supported withkidney histology finding, in which the PEG-DiDex treated mice were foundwith mainly mild/moderate nephritis and more normal glomeruli than Dexand saline groups (FIG. 5 ). It is worth noting of an extraordinaryfinding during the histological evaluation: signs of severe nephritiswere found in kidney sections from a NZW control mouse. NZB/W F1 is theoffspring of an NZB/B1NJ (Jackson Laboratory) female and an NZW/LacJ(Jackson Laboratory) male. Both inbred parental strains develop certainautoimmune abnormalities that are observed in the F1, but notnecessarily with similar onset or severity. The NZW/LacJ mice have anormal life span but do develop anti-dsDNA antibodies, high serum levelsof retroviral gp70 antigen, and nephritis later in life. Therefore, weattribute this isolated finding to the advanced age of the animal (38weeks).

Additional therapeutic benefit of PEG-DiDex treatment was also found inits capacity to more effectively attenuate systemic proinflammatorycytokine/chemokine levels than Dex treatment (FIG. 6 ).

This effective regulation of systemic proinflammatory cytokine/chemokinemay also explain the better-persevered bone quality in mice treated withPEG-DiDex (FIG. 7 ) than both Dex and saline groups, as severe systemicinflammation is known to be detrimental to the skeletal quality.Comparing to dose equivalent daily Dex treatment, the monthly PEG-DiDextreatment did not induce immune suppression as evident by WBC and totalserum IgG levels comparable to the saline group. Furthermore, micetreated monthly with PEG-DiDex were found with significantly higheradrenal gland mass than the dose equivalent daily Dex treated mice.Collectively, these data provide solid evidence of PEG-DiDex's superiorsafety profile to those of Dex.

PEG-DiDex also compares favorably with theN-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-based Dex prodrug(the “P-Dex”) disclosed previously (see, e.g., WO 2005/097073A1, CN101518654A, and CN 103059220A) on adverse effects. First, PEG-DiDex is aprodrug amphiphile that can self-assemble into micelles with the drugconjugated to chain terminus. The micelles disintegrate into monomersupon dilution in the circulation; whereas in P-Dex, the drug isconjugated to a polymer side chain. P-Dex does not form micelles andcannot disintegrate. Second, because of the use of relatively short PEG(˜2000 Da), PEG-DiDex is predicted to have a much shorter serumhalf-life than P-Dex, which constitutes its kidney targeting capacityand extremely low systemic toxicity as supported by the toxicity data.Third, the imaging results have shown that PEG-DiDex targets exclusivelynephritis, whereas P-Dex has high accumulation in liver and spleen inaddition to kidney. Moreover, when dPEG (a commercially available PEGwith a single molecular weight) is used, the PEG-DiDex should behavelike a small-molecule or biologic drug with a single molecular weight,without the polydispersity that polymer drug conjugates typically have.In addition, when Dex is conjugated to a multifunctional PEG carrier viaa hydrazone bond, the prodrug activation rate is slower than P-Dex.Therefore, by design the PEG-Dex hereby disclosed greatly reduces serumfree Dex concentration by limiting the prodrug's sequestration by WBCand its deposition in the liver and spleen; whereas the decreasedmolecular weight and serum half-life of P-Dex would lead to lower liverand spleen distribution.

Optical imaging-based in vivo biodistribution data (FIG. 8 ) indicatePEG-DiDex's dominant and sustained distribution organ in NZB/W F1 miceis the inflamed kidney. The distribution to other organs was verylimited, which is in stark comparison to the observation in mice treatedwith P-Dex (with high liver and spleen deposition, data not shown). Thisresult was further supported by PEG-DiDex's inability to reduce systemicanti-dsDNA level (FIG. 9 ) and to resolve splenomegaly (data not shown).These findings collectively suggest that the outstanding safety profileof PEG-DiDex may be attributed to its nephritis-oriented distributionpattern. In NZW control mice, PEG-DiDex's nephrotropic distributionpattern was repeated, but with the tissue prodrug concentration at amuch lower level. It indicates that the kidney retention of PEG-DiDex isinflammation selective.

Immunohistological and flow cytometry analyses revealed that Alexa Fluor488-labeled PEG-DiDex was mainly sequestered by CD11-b⁺ (monocytes),F4/80 (macrophages), CD146⁺ (endothelial cells) and CD326⁺ (epithelialcells) in the kidneys of NZB/W F1 mice. After 8 weeks of treatment, thekidney of PEG-DiDex treated NZB/W F1 mice showed a significantly lowermonocyte/macrophage population and local inflammatory cytokine levelthan dose equivalent Dex treated animals, suggesting the much effectiveand sustained local anti-inflammatory effect of PEG-DiDex. The prodrug'sinternalization, subcellular activation and inflammation resolution werefurther validated with in vitro cell culture systems.

In summary, the present invention provides a novel micelle-formingPEG-based dexamethasone prodrug (PEG-DiDex) with superior and sustainedefficacy against lupus nephritis, but without typical glucocorticoidside effects. While it is hardly perceivable that the Dex released fromPEG-DiDex would attenuate inflammation via a different molecularmechanism, this newly developed prodrug indeed altered the Dex'spharmacology on the physiology level by restricting the Dex'sdistribution to the inflamed kidney and providing a sustained localconcentration of Dex via the gradual activation of PEG-DiDex within theendosomal/lysosomal compartments. Given its outstanding therapeuticefficacy and safety profile, PEG-DiDex can provide better clinicalmanagement of lupus nephritis. Moreover, the novel glucocorticoidprodrugs may also find wider applications in other renal pathologies,such as minimal change disease, IgA nephropathy, focal segmentalglomerulosclerosis, and kidney transplant, since glucocorticoids arecommonly used in managing these conditions as well.

The following non-limiting Examples further illustrate certain aspectsof the present invention.

Examples Synthesis and Characterization of PEG-DiDex

Materials

Polyethylene glycol monomethyl ether 1900 (mPEG, 1.9 kDa) andN-Fmoc-L-glutamic acid were purchased from Alfa Aesar (MA, USA).Dexamethasone (Dex) was obtained from Tianjin Pharmaceuticals Group Co.,Ltd. (Tianjin, China). Dexamethasone phosphate was purchased fromHawkins, Inc. (Minneapolis, MN, USA). Dexamethasone phosphate disodiumwas purchased from BUFA (The Netherlands). Peperidine was purchased fromSigma-Aldrich (St. Louis, MO, USA). Dess-Martin periodinane was obtainedfrom Oakwood Products, Inc. (Estill, SC, USA). IRDye 800CW NHS ester waspurchased from LI-COR Biosciences (Lincoln, NE, USA). Alexa Fluor 488NHS ester was obtained from Life Technologies (Carlsbad, CA, USA).Sephadex LH-20 resins were purchased from GE HealthCare (Piscataway, NJ,USA). All solvents and other reagents if not specified were purchasedfrom Fisher Scientific or ACROS and used without further purification.

Instruments

¹H and ¹³C NMR spectra were recorded on a 500 MHz NMR spectrometer(Varian, Palo Alto, CA, USA). Electrospray ionization mass spectrometrywas performed on a LCQ Classic Mass Spectrometer (Finnigan MAT, SanJose, CA, USA). HPLC analyses were done on an Agilent 1100 HPLC system(Agilent Technologies, Inc., Santa Clara, CA, USA) with a reverse phaseC18 column (Agilent, ZORBAX 300SB-C18, 4.6×250 mm, 5 μm). In vivonear-infrared fluorescence (NIRF)-based optical imaging was accomplishedon a LI-COR Pearl™ Impulse Small Animal Imaging System (Lincoln, NE,USA). Bone qualities were analyzed using a high-resolution micro-CTsystem (Skyscan 1172, Skyscan, Aartselaar, Belgium). The averagehydrodynamic diameter, polydispersity index (PDI) and zeta potential ofmicelles were determined by dynamic light scattering (DLS) experimentsusing a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK).The micelles morphology was observed using a Tecnai G2 Spirittransmission electron microscope (TEM) (FEI, Hillsboro, OR, USA) at anacceleration voltage of 80 kV. Digital images were acquired using aKeenView high-resolution camera and analyzed using Soft ImagingSolutions AnalySIS ITEM digital software. The quantification offluorescence signal intensities of IRDye 800 CW, Alexa Fluor 488 andpyrene were measured using Spectramax M2 spectrofluorometer (MolecularDevices, Sunnyvale, CA). The flow cytometry analyses were performedusing a FACSCalibur flow cytometer (BD Biosciences). A Waters e2695system equipped with a Waters 2489 absorption detector and a Waters QtofMicro electrospray ionization mass spectrometer was used to perform highperfor-mance liquid chromatography/mass spectrometry analyses.

The Synthesis of Amphiphilic Macromolecular Dexamethasone Prodrug(PEG-DiDex)

PEG-DiDex, a polyethylene glycol (PEG)-based amphiphilic dexamethasoneprodrug, was successfully synthesized according to the route illustratedin Scheme 1. The identity of the polymeric prodrug and the absence offree Dex were confirmed using LC-MS/MS. The multi-step synthesis isstraightforward with high yield at each step. Due to the utility ofhydrazone as the prodrug activation trigger that links Dex to glutamicacid and the overall dimer design, at least 4 syn/anti hydrazoneconfigure isomers can be formed. These isomers of the Dex dimer(compound 6) were chromatographically separated using LC-MS/MS withchromatography conditions stated in the Instruments section. Massspectra (positive ion ESI) for these isomers showed the molecular ion[M+H]⁺ at 1066.7, which confirms their monoisotopic mass of 1065.7. Thetheoretical Dex content in PEG-DiDex is calculated as 26.7 wt %. Aftercomplete hydrolysis of the prodrug, the HPLC analysis showed that 26.4wt % of the prodrug was released in the form of intact dexamethasone,suggesting the PEG-DiDex prodrug micelle synthesized has a ˜99% purity.

As shown in Scheme 1, the prodrug was synthesized by conjugating a Dexdimer to polyethylene glycol (PEG) 2000 chain terminus via aglutamate/glycine/hydrazone linker system.

Synthesis of Compound 1

Dexamethasone (7.84 g, 20 mmol) and imidazole (2.72 g, 40 mmol) weredissolved in anhydrous DMF (40 mL) and the solution was cooled to 0° C.tert-butylmethylsilyl chloride (TBSCl, 3.3 g, 22 mmol) was added. Thesolution was stirred at 0° C. for 3 hours and then allowed to roomtemperature for 2 hours. Ethyl acetate (100 mL) was added and washedwith brine (80 mL×4). The organic phase was dried over MgSO₄ and thenthe solvent was removed. The residue was purified with flashchromatography (ethyl acetate/hexanes=1/2) to give 9.98 g of compound 1(98.5% yield).

¹H NMR (500 MHz, CDCl₃): δ (ppm)=7.26 (d, J=10.0, 1H), 6.32 (d, J=10.0,6.10 (s, 1H), 4.63 (d, J=18.0, 1H), 4.38 (d, J=18.0, 1H), 4.37 (m, 1H),3.23 (s, 1H), 3.05 (m, 1H), 2.62 (td, J=13.5, 6.0, 1H), 2.51 (s, 1H),2.38 (m, 3H), 2.22 (m, 1H), 1.82 (m, 1H), 1.75 (m, J=7.0, 1H), 1.56 (m,1H), 1.55 (s, 3H), 1.45 (d, J=13.5, 1H), 1.24 (m, 1H), 1.06 (s, 3H),0.92 (s, 9H), 0.91 (d, J=7.0, 3H), 0.103 (s, 3H), 0.098 (s, 3H).

¹³C NMR (125 MHz, CDCl₃): δ (ppm)=209.474, 186.768, 166.725, 152.226,129.617, 124.904, 91.147, 72.159, 71.853, 69.462, 48.503, 43.635,37.164, 36.146, 34.240, 34.084, 32.289, 31.045, 27.296, 25.814, 22.931,22.886, 18.442, 17.168, 14.810, −5.328, −5.4662.

Synthesis of Compound 2

The starting material 1 (2.53 g, 5 mmol) and NH₂NH₂ monohydrate (750 mg,15 mmol) were dissolved in methanol (25 mL). Acetic acid (60 mg, 1 mmol)was added and the solution was stirred at room temperature for 5 hours.Ethyl acetate (100 mL) was added and washed with brine (80 ml×4). Theorganic phase was dried over MgSO₄ and then the solvent was removed. Theresidue was purified with flash chromatography (ethylacetate/hexanes=1/1) to give 1.14 g of compound 2. A total of 1.24 gstarting material was recovered. The final yield was calculated as85.8%. Due to the formation of hydrazone bond, the product is a mixtureof two syn/anti hydrazone configure isomers, which can't be separated byflash chromatography. NMR analysis suggests the molar ratio of the twoisomers is 1.75:1.

¹H NMR (500 MHz, d₆-DMSO): δ (ppm)=6.66 (dd, J=10.2 Hz, 1.5 Hz), 6.34(s, J=10.2 Hz, 6.28 (dd, J=10.2 Hz, 1.9 Hz), 6.14 (d, J=10.2 Hz), 5.99(s), 5.24 (br, s), 4.61 (d, J=18.0 Hz), 4.59 (d, J=18.0 Hz), 4.38 (d,J=18.0 Hz), 4.36 (m), 3.05 (m, 1H), 3.03 (s), 2.97 (t, J=4.8 Hz), 2.62(td, J=13.5, 5.8 Hz), 2.51 (td, J=13.5, 5.8 Hz), 2.40-2.20 (m),1.70-1.60 (m), 1.55-1.50 (m), 1.476 (s), 1.472 (s), 1.40 (s), 1.37 (s),1.24-1.20 (m), 1.04 (s), 0.92 (s), 0.90 (d, J=7.3 Hz), 0.103 (s). MS(ESI): m/z=521.5 (M+H⁺), calculated: 520.3.

Synthesis of Compound 3

Compound 2 (2.86 g, 5.5 mmol), dimethylaminopyridine (DMAP, 201 mg, 1.65mmol) were dissolved in anhydrous DMF (15 mL) and the solution wascooled to 0° C. Then Fmoc-glycine (2.12 g, 7.15 mmol),dicyclohexylcarbodiimide (DCC, 1.70 g, 8.25 mmol) were added to thesolution. The solution was stirred at 0° C. for 3 hours. Ethyl acetate(100 mL) was added and washed with brine (80 mL×4). The organic phasewas dried over MgSO₄ and then the solvent was removed. The residue waspurified with flash chromatography (ethyl acetate/hexanes=1:1) to give3.72 g of compound 3 (84.5% yield).

Synthesis of Compound 4

Compound 3 (3.0 g, 3.75 mmol) was dissolved in dichloromethane (DCM, 10mL). The solution was cooled to 0° C. with ice-water bath. Piperidine (1mL) was added. The solution was stirred at 0° C. for 1 hour. Ethylacetate (100 mL) was added and washed with brine (80 mL×3). The organicphase was dried over MgSO₄ and then the solvent was removed. Toluene (50mL) was added and then evaporated to remove the residue piperidine. Theresidue was then purified with flash chromatography (ethyl acetate andthen ethyl acetate/methanol=2.5/1) to give 1.96 g of compound 4 (90.6%yield).

¹H NMR (500 MHz, d₆-DMSO): δ (ppm)=7.01 (d, J=10.2 Hz), 6.83 (d, J=10.2Hz), 6.77 (s), 6.66 (d, J=10.2 Hz), 6.57 (d, J=10.2 Hz), 6.56 (s), 6.43(d, J=10.2 Hz), 6.38 (d, J=9.8 Hz), 6.27 (d, J=10.2 Hz), 6.18 (d, J=9.8Hz), 6.00 (s), 5.92 (s), 5.15 (s), 5.13 (s), 4.94 (s), 4.76 (d, J=9.0Hz), 4.27 (d, J=9.0 Hz), 4.11 (br), 2.88 (br), 2.70-2.50 (m), 2.49 (s),2.40-2.20 (m), 2.15-2.05 (m), 1.73-1.63 (m), 1.63-1.53 (m), 1.41 (s),1.40 (s), 1.37 (s), 1.35-1.25 (m), 1.10-1.00 (m), 0.87 (s), 0.84 (s),0.76 (d, J=6.8 Hz), 0.03 (s), 0.02 (s). MS (ESI): m/z=578.3 (M+H⁺),calculated: 577.3.

Synthesis of Compound 5

Compound 4 (444 mg, 0.768 mmol) was dissolved in anhydrous DMF (3 mL),Fmoc-glutamic acid (135 mg, 0.366 mmol), DCC (226 mg, 1.098 mmol) andhydroxybenzotriazole (HOBt, 148 mg, 1.098 mmol) were added. The solutionwas stirred at room temperature for 4 hours. Ethyl acetate (100 mL) wasadded and washed with brine (80 mL×3). The organic phase was dried overMgSO₄ and then the solvent was removed. The residue was then purifiedwith flash chromatography (ethyl acetate/methanol=10/1) to give 471 mgof compound 5 (86.5% yield). Due to the presence of multiplesyn/anti-hydrazone groups in the Dex dimer, the assignment of peaks inthe ¹H NMR spectrum of compound 5 was complex, so LC-MS/MS was used toconfirm the identity of compound 5.

Synthesis of Compound 6

Compound 5 (450 mg, 0.3 mmol) was dissolved in DCM (4.5 mL). Thesolution was cooled to 0° C. with ice-water bath. Piperidine (1.5 mL)was added. The solution was stirred at 0° C. for 1 hour. Ethyl acetate(100 mL) was added and washed with brine (80 mL×3). The organic phasewas dried over MgSO₄ and then the solvent was removed. The residue wasthen purified with flash chromatography (ethyl acetate and followed byethyl acetate/methanol=3/1) to give 330 mg of compound 6 (86.4% yield).MS (ESI): m/z=1266.7 (M+H⁺), calculated: 1265.7.

Synthesis of Compound 7 (PEG-DiDex)

mPEG-COOH (100 mg, 0.052 mmol), HOBt (70.2 mg, 0.52 mmol) and DCC (107mg, 0.52 mmol) were dissolved in DMF (3 mL) and the solution was stirredat room temperature for 1 hour. Compound 6 (428 mg, 0.34 mmol) wasadded. The solution was stirred at room temperature for 24 hours andthen applied to LH-20 column to separate the polymeric fraction. Afterevaporation of the solvent, the residue was dissolved intetra-n-butylammonium fluoride (TBAF, 1 M, 2 mL). The solution wasstirred for 2 hours. The resulting solution was again applied onto LH-20column to give 118.7 mg of compound 7 (PEG-DiDex, 77.8% yield).

MS (ESI): two clusters of peaks were observed in the mass spectrum: oneappears at m/z≈1550, representing the diion peaks of PEG-DiDex; and theother at m/z≈1100, representing triion peaks of PEG-DiDex. Theappearance of multiple peaks at each m/z value can be attributed to thepolydispersity of mPEG used. For example, the peak at 1546.7 representsdiionic peak of PEG-DiDex with 44 repeating ethylene glycol unit inmPEG; the peak at 1068.5 represents triionic peak PEG-DiDex with 46repeating ethylene glycol units in mPEG.

Synthesis of Symmetric mPEG-(Dex-Dimer) Compound 9

Synthesis of Compound 8

The monomethyl citrate (0.206 g, 1 mmol) is dissolved in anhydrous DMF(15 mL) and the solution is cooled to 0° C. DCC (0.494 g, 2.4 mmol),compound 2 (1.144 g, 2.2 mmol) and DMAP (48.8 mg, 0.4 mmol) are added.The solution is stirred at 0° C. for 3 hours. Ethyl acetate (100 mL) isadded and washed with brine (80 mL×4). The organic phase is dried overMgSO₄ and then the solvent is removed. The residue is purified withcolumn chromatography (ethyl acetate/hexanes=1/1) to give compound 8.

Synthesis of Compound 9

Compound 3 (0.9 g, 0.75 mmol) and mPEG-NH₂ (0.285 g, 0.15 mmol) aredissolved in anhydrous DMF (5 mL) and the solution is heated to 80° C.for 6 hours under the protection of Argon. Then the solution is allowedto room temperature and TBAF (3 mL, 1 M in THF) is added. The solutionwas stirred for 1 hour at room temperature. The solution is thenconcentrated and then purified by LH-20 chromatography to give Compound9.

Synthesis of Symmetric mPEG-(Dex-Trimer) Compound 11

Synthesis of Compound 10

The Pentaerythritol derivative (0.41 g, 1 mmol) is dissolved inanhydrous DMF (5 mL). DCC (0.82 g, 4 mmol), HOBt (0.405 g, 3 mmol) andEt₃N (0.30 g, 3 mmol) are added and the solution is stirred for 30 min,and mPEG-NH₂ (0.38 g, 0.2 mmol) is added. The solution is stirred atroom temperature for 20 hours. It is then purified by LH-20 to givecompound 10.

Synthesis of Compound 11

Compound 10 (0.23 g, 0.1 mmol) and sodium hydroxide (12 mg, 0.3 mmol)are dissolved in a mixture solution of methanol and water (1:1, 5 mL).The solution is stirred at room temperature for 5 hours. Then HCl (3 mL,1M) is added. The solvent is then removed and the residue is purified byLH-20 to give the deprotected intermediate. It is then dissolved inanhydrous dichloromethane (5 mL). DCC (0.30 g, 1.5 mmol), HOBt (0.20 g,1.5 mmol) and Et₃N (0.15 g, 1.5 mmol) are added and the solution isstirred for 30 min, then compound 2 (0.62 g, 1.2 mmol) is added. Thesolution is stirred at room temperature for 20 hours. The solution isthen loaded on to a silica gel column and ethyl acetate/hexanes=1:1 isused as eluent to recover the unreacted compound 2. Then methanol isused as eluent to get the crude product. The solvent is then removed andthe residue is dissolved in THE (5 mL) and TBAF (1.5 mL, 1 M in THF) isadded. The solution is stirred at room temperature for 1 hour. Thesolution is then concentrated and purified by LH-20 to give the finalproduct compound 11.

Synthesis of mPEG-Dex, Compound 12

Synthesis of Compound 12

mPEG-CH₂COOH (0.19 g, 0.1 mmol) was dissolved in anhydrous DMF (5 mL).DCC (0.21 g, 1 mmol), HOBt (0.135 g, 1 mmol) and Et₃N (0.1 g, 1 mmol)were added and the solution was stirred for 30 min, and then thecompound 2 (0.26 g, 0.5 mmol) was added. The solution was stirred atroom temperature for 20 hours. TBAF (0.5 mL, 1 M in THF) was added andthe solution was stirred for 1 hour, followed by LH-20 purification togive compound 12.

Synthesis of Symmetric Dex-PEG-Dex Compound 13

Synthesis of Compound 13

HOOCCH₂-PEG-CH₂COOH (0.14 g, 0.07 mmol) was dissolved in anhydrous DMF(5 mL). DCC (0.142 g, 0.7 mmol), HOBt (0.093 g, 0.7 mmol) were added andthe solution was stirred for 30 min, and then the compound 2 (0.398 g,0.7 mmol) was added. The solution was stirred at room temperature for 15hours. TBAF (1 mL, 1 M in THF) was added and the solution was stirredfor 1 h and then purified by LH-20 to give compound 13.

Synthesis of Asymmetric mPEG-(Dex-Tetramer) Compound 15

Synthesis of Compound 14

Fmoc-L-glutamic acid (93 mg, 0.25 mmol) is dissolved in anhydrous DMF (5mL). DCC (0.62 g, 3 mmol), HOBt (0.41 g, 3 mmol) are added and thesolution is stirred for 30 min, and then compound 6 (0.79 g, 0.625 mmol)is added. The solution is stirred at room temperature for 15 hours.Ethyl acetate (100 mL) is added and washed with brine (80 mL×4). Theorganic phase is dried over MgSO₄ and then the solvent was removed. Theresidue is purified with flash chromatography (ethylacetate/methanol=5/1) to give the product which is dissolved indichloromethane (3 mL), piperidine (1 mL) is then added and the solutionis stirred for 1 hour and then the solution is purified by columnchromatography (ethyl acetate/methanol=2.5/1) to give compound 14.

Synthesis of Compound 15

The mPEG-CH₂COOH (50 mg, 0.025 mmol) is dissolved in anhydrous DMF (3mL). DCC (0.10 g, 0.5 mmol), HOBt (68 mg, 0.5 mmol) are added and thesolution is stirred for 30 min, and then the compound 14 (0.43 g, 0.15mmol) is added. The solution is stirred at room temperature for 15hours. TBAF (0.5 mL, 1 M in THF) is added and the solution is stirredfor 1 hour and then the solution is purified by LH-20 to give compound15.

Characterization and Testing of PEG-Dex Dimer Prodrug (PEG-DiDex)Formation of PEG-DiDex Micelles

With an amphiphilic structural design, PEG-DiDex can form micellesthrough self-assembly. PEG-DiDex (26.5 mg) was dissolved in distilledwater (1 mL) and equilibrated at 37° C. for 4 hours to allow micellesformation. The micelle solution was then diluted to 3 mg/mL (1.02×10⁻³M) by adding double distilled water and then used for the followingcharacterization.

Micelle Characterization

The average hydrodynamic diameter, polydispersity index (PDI) andζ-potential of the micelles were determined by dynamic light scattering(DLS) using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire,UK). The intensity of scattered light was measured at a 1730 scatteringangle. To understand the micelles morphology, transmission electronicmicroscopy (TEM) was used to visualize micelles deposited onformvar/silicone monoxide coated 200 mesh copper grids surface.

Pyrene-based fluorescence polarization method was used to determine thecritical micelle concentration (CMC) for PEG-DiDex. For samplepreparation, known amounts of stock solution of pyrene in acetone wereadded to empty wells of 96-well plate. Aqueous solutions of PEG-DiDex atdifferent concentrations were added to the wells and evaporate for 2hours at room temperature. The pyrene concentration in the finalsolution was 0.6 μM, which is slightly below its water solubility inwater at room temperature. Before measurement, the samples from eachwell were transferred to a quartz 96-well plate, and the fluorescenceintensity was measured with excitation wavelength of 334 nm and emissionwavelength at 373 nm (I1) and 384 nm (I3) using a fluorescencemicroplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). Ratioof fluorescence intensity I1/I3 was plotted against prodrugconcentration to obtain the CMC value.

To understand the impact of pH values and the presence of serum proteinson the release of Dex from PEG-DiDex, the prodrug (3 mg/mL) wasdissolved in acetate buffers (pH=5.0, 6.5 and 7.4) and mouse serum (0.2wt % sodium azide as bacteriostat). Pluoronic F127 was added as in orderto create sink condition. The micelle solutions were placed in a shakingincubator (60 r/min) at 37° C. At selected time points, the releasingsolution (0.5 mL) was withdrawn, neutralized with NaOH (0.1 M) andanalyzed with HPLC to determine the free Dex concentration. The analysisof each sample was performed in triplicate. The accumulative release ofDex from PEG-DiDex micelles was calculated according the followingequation, where C; refers to the concentration of Dex at time i.

${{Accumulative}{release}\left( {{wt}\%} \right)} = {\frac{{V_{i} \times C_{i}} + {0.5 \times {\sum}_{i = 1}^{n - 1}C_{i}}}{{weight}{of}{Dex}{in}{the}{micells}} \times 100\%}$

To quantify the Dex content in PEG-DiDex, the prodrug (1 mg) wasdissolved in HCl (0.5 mL, 0.1 N) overnight. The sample (50 μL) waswithdrawn and neutralized by addition of NaOH (50 μL, 0.1N), thendiluted in acetonitrile (CAN, 0.9 mL). The sample (in triplet) wasanalyzed using an Agilent 1100 HPLC system equipped with a reverse phaseC18 column (Agilent, ZORBAX 300SB-C18, 4.6×250 mm, 5 μm). Mobile phase:acetonitrile/water=30/70; detection wavelength, 240 nm; flow rate, 1mL/min; Injection volume, 10 μL. The Dex content in PEG-DiDex was thencalculated based on the HPLC analysis result.

Testing Results

Dynamic light scattering (DLS) measurement indicates that the PEG-DiDexcan indeed form micelle with an average micelle diameter of ˜11 nm, apolydispersity index (PDI) of 0.345 and a ζ-potential of −5±4.61 mV. Asshown in the TEM images (FIG. 2 ), the micelles deposited on thesubstrate showed an average ˜30 nm diameter. The size discrepancy withthe DLS measurement may be attributed to the collapse of the micellesduring the sample preparation process. Using pyrene-based fluorescencepolarization method, the critical micelle concentration (CMC) value ofPEG-DiDex was determined to be 2.5×10⁻⁴ M.

As the Dex activation trigger, the hydrazone bond in the PEG-DiDexdesign can only be cleaved via acidic environment. This was confirmed bythe in vitro drug release experiment. As shown in FIG. 3 , at pH=5.0 inacetate buffer, 2% of the Dex loading was released within the first twodays. It was then followed by a sustained release at roughly 0.27%/dayfor the next 26 days.

Treating Lupus Nephritis with PEG-DiDex

Beginning at 20 weeks of age, NZB/W F1 female mice (JacksonLaboratories, Bar Harbor, ME) were randomized into three test groups(saline control, Dex and PEG-DiDex). Their urine protein level wasmonitored weekly using Albustix Reagent Strips (Siemens Healthineers).Only mice with established nephritis, as evidenced by sustainedalbuminuria (≥100 mg/dL) over 2 weeks, were enrolled in the study.PEG-DiDex treatment (106 mg/kg, containing 28 mg/kg of dexamethasone,n=10) and saline (n=12) were administered as a monthly i.v. injection.The Dex treatment (dexamethasone 21-phosphate disodium, 1.32 mg/kg,containing 1.00 mg/kg of dexamethasone, n=11) was given as daily i.v.injection. All treatments continued for 8 weeks. The body weight andproteinuria level of the animals was monitored on a weekly basis.Peripheral blood was collected from saphenous vein every 4 weeks forserum analyses. Mice that developed severe proteinuria (≥2000 mg/dl) orshowed signs of distress (e.g. reduced mobility, weight loss >20%,edema, unkempt appearance) were sacrificed immediately. The survivingmice were monitored for an additional two weeks after the lasttreatment. The mice were then euthanized by CO₂ asphyxiation, with allmajor tissues and organs isolated, weighted and processed at necropsy.All animal procedures were approved by the Institutional Animal Care andUse Committee (IACUC) of University of Nebraska Medical Center (UNMC).

Analysis of Bone Quality

Femoral bone quality was analyzed using a Skyscan 1172 micro-CT system.The micro-CT scanning parameters were set as the following: voltage 48kV, current 187 μA, exposure time 620 msec, resolution 6.07 μm, andaluminum filter 0.5 mm. Three-dimensional reconstructions were performedwith NRecon and DataViewer software (SkyScan). Trabecular bone wasselected for analysis based on a polygonal region of interest within thecenter of the femur, starting at 20 slices (0.25 mm) proximal from thegrowth plate and extending proximally 80 slices (0.99 mm) further.Trabecular bone volume/tissue volume (BV/TV), the mean bone mineraldensity (BMD), trabecular number and thickness were quantified with CTAnsoftware (SkyScan).

Near-Infrared Imaging Study

After the proteinuria was established, NZB/W F1 mice (n=6) were givenIRDye 800 CW-labeled PEG-DiDex (IRDye 800 CW dose at 148 nmol/kg, Dexequivalent dose of 28 mg/kg) via tail vein injection. The same dose ofIRDye 800 CW-labeled PEG-DiDex was also given intravenously to NZW mice(n=6, healthy control). At selected time points (1 and 4 days postadministration), the mice were euthanized and perfused with saline. Allmajor organs (i.e. heart, lung, liver, spleen, kidney and adrenal gland)were isolated and imaged using a LI-COR Pearl™ Impulse Small AnimalImaging System to evaluate the distribution and retention of PEG-DiDex.

Flow Cytometry Analysis

After the proteinuria was established, NZB/W F1 mice (n=6) and NZW mice(healthy control) were given Alexa Fluor 488-labeled PEG-DiDex (AlexaFluor 488 dose at 300 nmol/kg, Dex equivalent dose of 28 mg/kg) via tailvein injection. At selected time points (1 and 4 days postadministration), the animals were euthanized and perfused. White bloodcells were isolated from peripheral blood. Bone marrow, kidney, spleenand liver were harvested, macerated, and passed through a 70-μm strainerto prepare single-cell suspensions. Cells were marked by the followingantibodies: PE-labeled anti-mouse CD3e (17A2), CD11b, F4/80, NK1.1,CD146, prominin (Miltenyi Biotec) and CD19 (eBioscience Inc.);APC-labeled anti-mouse CD11c, Ly-6G, CD326, CD117; anti-mouseGL7-eFluor660. The cells were analyzed using a FACSCalibur flowcytometer (BD Biosciences).

Statistics

Most of the statistical analyses were performed using SPSS software(version 19.0). The data that does not assume to follow a normaldistribution was compared using the Kruskal-Wallis test, a nonparametricalternative to one-way analysis of variance. To evaluate specificdifferences between experimental groups, Tukey's post hoc test andMann-Whitney U test were used in the comparisons of normal-distributedand non-normal distributed data respectively. Two-tailed P values ≤0.05were considered significant.

For inflammatory cytokine/chemokine analysis, the data obtained were log2 transformed to make them more normally distributed. The mixed effectsmodel with AR(1) correlation among repeated measures over time of thesame animal were used to fit the log transformed cytokine expressionvalues for each cytokine separately. Three different comparisons wereconducted to evaluate (1) difference between the treatment groups ateach observation time; (2) difference between different times withineach treatment group; and (3) difference in the change of the cytokinesexpression from baseline among different treatment groups. TheBenjamini-Hochberg method was used to control the false discovery ratefor multiple comparisons. The results on the differences at log-scaleddata, the standard error, the raw p value without adjustment for falsediscovery rate, and the adjusted p value for BH false discovery ratecontrol were reported.

PEG-DiDex Effectively Ameliorated Proteinuria and Improved the Survivalof NZB/W F1 Mice with Severe Nephritis

To evaluate the therapeutic potential of PEG-DiDex, it was given wasadministered monthly to NZB/W F1 female mice (˜28 wks) with fullydeveloped developed nephritis, as evidenced by sustained albuminuria.The treatment was continued for 8 weeks. Dose equivalent daily Dextreatment and monthly saline administration were used as controls. Asshown in FIG. 4A, 2 months PEG-DiDex treatment result in albuminuriaresolution in 60% of the mice tested. For the daily Dex treatment group,only 18% of resolution was achieved at the end of the experiment. Overthe entire experimental time course, albuminuria persisted in 100% ofthe mice of the saline group. A total of 42% of mice in saline grouphave to be euthanized due to severe nephritis (FIG. 4B), as mandated byIACUC protocol. This observation is in agreement with others' finding ofthe median survival age of NZB/W F1 mice to be around 36 wks. Whiledaily Dex treatment improved the mice survival to ˜82%, all animalssurvived at the end of the experiment in the PEG-DiDex treated group,suggesting a superior therapeutic efficacy than daily Dex treatment.

For additional evidence of PEG-DiDex's superior therapeutic efficacy,the kidneys from the tested animals were further sectioned and stainedwith PSA. They were then examined by a pathologist (KWF), who was blindto the group design. The periodic-acid schiff (PAS) stained kidneysections were also graded by a histopathologic score system with a4-point scale. As shown in FIG. 5 , more than 40% of the mice from Dexand saline group showed histological evidence of severeglomerulonephritis (scored 3 and 4 points) typified by wire-looplesions, acute tubular necrosis (ATN), glomerular scarring, cellularcrescents and hyaline thrombi formation. In contrast, in PEG-DiDextreated group, only ˜11% of the mice were graded as severeglomerulonephritis, which was significantly lower than that in salineand Dex groups. Histological abnormities were observed in 26% glomeruliin PEG-DiDex group, which is close to the frequency (21.6%) found in theNZW mice. Comparing to this observation, 40% and 52% glomeruli in Dexand saline groups were found to be abnormal respectively, which furthersupport the superior therapeutic efficacy of PEG-DiDex in treating lupusnephritis.

PEG-DiDex Treatment Attenuates Tissue-Damaging ProinflammatoryCytokines/Chemokines.

As shown in FIG. 6 , only animals treated with PEG-DiDex causedstatistically significant reduction in MCP-1, IFN-β, and IFN-γ values atthe end of 2-month treatment. Dex treatment, on the other hand, did notinduce the improvement.

PEG-DiDex Treatment does not Lead to Typical GC Toxicities

To understand the impact of PEG-DiDex treatment on bones, the femoralmean bone mineral density (BMD) and micro-architecture were evaluatedusing a high resolution μ-CT (Skyscan 1172). The BMD value andtrabecular thickness in the femoral trabecular bone of PEG-DiDex treatedmice were significantly higher than that observed in both saline and Dexgroup (FIG. 7A, C; P<0.05). A trend of increase in trabecular bonevolume/tissue volume (BV/TV) value was also observed, though suchincrease is not statistically significant (FIG. 7B, P >0.05). Comparingto NZW mice (as a healthy control), even the saline group exhibitssignificantly lower values of BMD, BV/TV and trabecular thickness (datanot shown), which suggest the systemic inflammatory condition of NZB/WF1 mice is detrimental to the skeletal quality. The PEG-DiDex treatmentnot only avoided the negative GC impact on the bone, but also furtherimpeded the inflammation-associated skeletal deterioration by effectiveamelioration of nephritis.

Chronic exposure to GC therapy is known to be associated with systemicimmunosuppression. To understand if PEG-DiDex as a GC prodrug would besimilarly immune suppressive, we evaluated the end point total serum IgGlevel and the peripheral white blood cell (WBC) counts during the timecourse of the experimental. As shown in FIG. 7D, PEG-DiDex treated miceexhibited a WBC counts similar to the saline group, but at asignificantly higher value than the Dex treated group (P<0.05). Also seein FIG. 7E, PEG-DiDex monthly administration did not alter serum IgGlevel during the course of the treatment, while the animals treated withdaily Dex had a significant drop of serum IgG value after 1 month oftreatment (P<0.05). These data collectively suggest the absences ofsigns of immune suppression in animals treated with PEG-DiDex.

GC exposure, even in short term may suppresshypothalamic-pituitary-adrenal (HPA) axis, leading to clinical atrophyof the adrenal gland. To understand if PEG-DiDex treatment would causeadrenal gland atrophy, we analyzed the end point adrenal gland mass. Themean adrenal gland mass in the Dex group was significantly lower thanthe PEG-DiDex group (FIG. 7F; P<0.05). There was no significantdifference in adrenal gland mass between the PEG-DiDex and saline groups(FIG. 7F; P >0.05). These data suggest that treatment by PEG-DiDex wouldnot induce adrenal gland atrophy.

PEG-DiDex Passively Targeted to Nephritic Kidneys in NZB/W F1 Mice

To understand the potentiated therapeutic efficacy and greatly reducedGC-associated toxicities of PEG-DiDex (a GC prodrug), the in vivobiodistribution of PEG-DiDex was analyzed using near-infrared opticalimaging. Both NZB/W F1 mice and NZW mice received intravenous injectionsof IRDye 800 CW-labeled PEG-DiDex. The animals were sacrificed on day 1and day 4 post injection and all vital organs (i.e. heart, lung, liver,spleen, kidney and adrenal gland) were harvested for the opticalimaging. As shown in FIG. 8 , in NZB/W F1 mice, the IRDye-labeledPEG-DiDex primarily accumulated in the kidneys and can be retained therefor at least 4 days. For NZW mice, PEG-DiDex was found to accumulate inkidneys, but the signal intensity of the retained prodrug was at a muchlower level especially at 4 days post administration, suggesting theinflammatory condition of the kidneys may especially facilitate thepassive targeting and retention of the prodrug in nephritis.

PEG-DiDex Treatment does not Alter Serum Anti-dsDNA Level

GCs are known to exert its therapeutic effect against lupus partiallythrough down regulation of anti-dsDNA antibody level. Therefore, itwould be of great interest to see if a Dex prodrug, such as PEG-DiDex,treatment would ameliorate lupus symptoms according to a similarpharmacology. As shown in FIG. 9 , daily Dex treatment was found tosignificantly reduce serum anti-dsDNA IgG levels at both 4 and 8 weekspost-treatment initiation. For saline and PEG-DiDex treatments, however,no significant impact on the anti-dsDNA IgG level was observed at eithertime point.

Characterization of PEG-DiDex and Drug Loading Efficiency

Effective hydrodynamic diameters (D_(eff)) and polydispersity index(PDI) of PEG-DiDex dimer were measured by dynamic light scattering (DLS)using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK).PEG-DiDex dimer was dissolved into PBS (pH 7.4) at a concentration of 2mg/mL. The DLS analysis showed the hydrodynamic size for PEG-DiDex dimerwas 273.5 nm with a PDI value of 0.464.

To quantify Dex loading in PEG-DiDex, 1 mg of of the prodrug wasdissolved in 0.5 ml of buffer (HCL, 0.1N) overnight. 50 μl of the samplewas neutralized by 50 ul NaOH (0.1N), and then diluted in 0.9 ml ACN. 1ml of the sample (triplet) was analyzed with an Agilent 1100 HPLC systemwith a quaternary pump (with degasser), an autosampler and a diode-arraybased UV detector. Mobile phase, acetonitrile/water=30/70; Detection, UV240 nm; Flow rate, 1 ml/min; Injection volume, 10 μl. The mean value andstandard deviation were obtained with Microsoft Excel. The HPLC analysisshowed that 26.38% of the prodrug was released as the form ofdexamethasone. Since the theoretical drug content of PEG-DiDex is26.75%, 98.6% of theoretical Dex content was covalently conjugated tothe prodrug.

PEG-DiDex Dimer Treatment of Lupus Nephritis Experimental Animals andDrug Treatment

Beginning at 28 weeks of age, (NZB×NZW)F1 female mice (JacksonLaboratories, Bar Harbor, ME) groups of mice were randomized intosaline, Dex and PEG-DiDex groups and monitored weekly for albuminuriausing Albustix dipsticks (Siemens Corp., Washington DC). Only the micein each group with established nephritis, evidenced by sustainedalbuminuria (≥100 mg/dl) over a monitoring period of 2 weeks, wereofficially enrolled in the study. PEG-DiDex (106 mg/kg, containing 28mg/kg of dexamethasone, n=10) and saline groups (n=12) were administeredvia a single injection per month. The free Dex group was given dailyi.v. injections of dexamethasone 21-phosphate disodium (n=11, Dex, 1.32mg/kg, containing 1.00 mg/kg of dexamethasone, Hawkins, Inc.,Minneapolis, MN). Treatment continued for 8 weeks. Mice were monitoredfor an additional two weeks after cessation of treatment. After that,the mice were euthanized and the spleen, kidneys, adrenal glands andleft femur were harvest.

PEG-DiDex Reverses Established Albuminuria, Extends Survival Rates andReduces Incidence of Severe Nephritis

To determine if PEG-DiDex could ameliorate established nephritis,PEG-DiDex was administered monthly to (NZB×NZW)F1 females beginning at˜28 weeks of age, after they had developed nephritis, as evidenced bysustained albuminuria. Treatment was continued for 8 weeks. Two controlgroups, one receiving dose equivalent daily Dex and the other receivinga monthly dose of saline, were also treated for 8 weeks. Mice weremonitored for an additional two weeks after cessation of treatment. Overthe entire experimental time course, albuminuria not only persisted in100% of the mice in the saline treated group, but also increased inseverity in most of these mice (75%) (FIG. 10A). However, in the Dexgroup, albuminuria was detected in 82% of the mice, and intensified injust 36% of the Dex treated mice, indicating that Dex treatment couldprevent progression of renal dysfunction.

By contrast, albuminuria resolved in 60% of the mice in the PEG-DiDexgroup (FIG. 10A), and 20% of the PEG-DiDex treated mice even showednegative albuminuria and thoroughly recovered. The fraction of mice inthe PEG-DiDex group that showed resolution of albuminuria wassignificantly greater than that in the Dex treated group, indicatingthat PEG-DiDex is more effective than dose equivalent Dex in resolvingalbuminuria associated with lupus nephritis.

Prior to the end of the experiment, ˜42% of mice in the saline group and˜18% of Dex treated mice were euthanized due to severe nephritis (FIG.10B). All mice in the PEG-DiDex group survived during the entiretreatment period, indicating that PEG-DiDex treatment increased thefraction of mice surviving until the end of the treatment period. Thesedata indicate that PEG-DiDex can extend the lifespan of (NZB×NZW)F1mice.

Evaluation of Treatment-Induced Side Effects

PEG-DiDex Treatment does not Affect Bone Quality

Peripheral dual x-ray absorptiometry was performed with a scanning speedof 20 mm/second and resolution of 0.2×0.2 mm. The micro-CT scanningparameters were as follows: voltage 48 kV, current 187 μA, exposure time620 msec, resolution 6.07 μm, and aluminum filter 0.5 mm.Three-dimensional reconstructions were performed with NRecon andDataViewer software (SkyScan). Trabecular bone was selected for analysisbased on a polygonal region of interest within the center of the femur,starting at 20 slices (0.25 mm) proximal from the growth plate andextending proximally 80 slices (0.99 mm) further. Trabecular bonevolume/tissue volume (BV/TV), the mean bone mineral density (BMD),trabecular number, and trabecular thickness were quantified with CTAnsoftware (SkyScan).

Osteoporosis is a major adverse side effect of long-term use of GCs. Toinvestigate the impact of PEG-DiDex on the skeleton, the femoral BMD andmicro-architecture were evaluated. Trabecular bone volume/tissue volume(BV/TV) and trabecular thickness did not differ significantly from themeans in the saline group (FIG. 7B, C; P >0.05), indicating thatPEG-DiDex did not negatively affect BV/TV or trabecular thickness of thebone. The mean bone mineral density (BMD) and trabecular number in thefemurs of PEG-DiDex treated mice were significantly higher than thatobserved in both saline and Dex group (FIG. 7A, D; P<0.05). Compare toNZW mice (as a healthy control), even the saline group exhibitssignificantly lower values of BMD, BV/TV and trabecular number (data notshown). Thus, PEG-DiDex treatment not only avoided osteoporosis inducedby long-term administration of GCs, but also ameliorated bone lesioncaused by other complications of SLE, rheumatoid arthritis, for example.

PEG-DiDex Treatment does not Lead to Peripheral White Blood CellsReduction

GC therapy is associated with immunosuppression. Therefore, we monitoredperipheral white blood cell (WBC) counts and during the experimentaltime course. In comparison of healthy control mice, peripheral whiteblood cell (WBC) counts were significantly lower in the other threegroups (data not shown). But the PEG-DiDex group exhibited significantlygreater WBC counts compared to the Dex group (FIG. 7D; P<0.05). The WBCcounts of Dex group were lower than the saline group, even though therewas no significant difference. The WBC counts did not differsignificantly between PEG-DiDex and saline groups. Thus, PEG-DiDextreatment ameliorates peripheral WBC reduction induced by GC therapy.

PEG-DiDex Treatment does not Induce Adrenal Gland Atrophy

GC therapy causes suppression of hypothalamic-pituitary-adrenal (HPA)axis and atrophy of the adrenal glands. Therefore, at necropsy, wedetermined the mass of the adrenal glands in each mouse. The meanadrenal mass in the Dex group was significantly lower than the PEG-DiDexgroup (FIG. 7F; P<0.05). There was no significant difference in adrenalgland mass between the PEG-DiDex and saline groups (FIG. 7F; P >0.05).These data suggest that treatment with PEG-DiDex did not induce adrenalgland atrophy.

In summary, PEG-DiDex treatment would potentiate lupus nephritisresolution in terms of prolonged lifespan and reduced incidence ofsevere nephritis, as well as a reduced risk of systemic toxicities andside effects.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as claimed in the appending claims.

All publications, patents, and patent applications cited in thisapplication are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated herein byreference.

1-21. (canceled)
 22. A method of preparing a glucocorticoid prodrugcompound of formula (XII):

or a pharmaceutically acceptable salt thereof; wherein: n is an integerselected from 10 to 300; m is 1, 2, 3, or 4; w is 1 or 2; GC is a moietyof a glucocorticoid drug molecule; A is absent or C₁-C₆ alkylene; B isabsent, NH, or C(O); D is absent, NH, O, or C(O); E is a linker selectedfrom:

 where i and j are independently 0 or an integer selected from 1 to 5; Gis NH; P is absent or C(O); Q is absent or C₁-C₆ alkylene; T is absentor C(O); X is absent, O, or NH; Y is absent, C(O), or C₁-C₆ alkylene; Zis absent, NR⁴, O, C₁-C₆ alkylene, or a linker comprising a branchedstructure capable of connecting to one or more glucocorticoid drugmolecular moieties, said linker optionally comprising one or moreheteroatoms independently selected from O, S, and N; and R¹ is H, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, 5-10 memberedheteroaryl, or 5-10 membered heterocyclyl, each group except Hoptionally substituted; R⁴ at each occurrence is independently H orC₁-C₄ alkyl; wherein when any of A, B, D, E, G, P, Q, T, X, Y, and Zgroups is absent, its two available adjacent groups are single-bonded toeach other directly, the method comprising steps of: (a1) coupling alinker compound comprising a moiety of structure E with a glucocorticoidhydrazone derivative of formula (A) or (B) to form an intermediate; and(b1) coupling the intermediate with a polyethylene glycol derivativeselected from PEG-OH, PEG-NH₂, PEG-CO₂H, and PEG-CO₂R¹⁰ to form aprecursor compound to the compound of structure (XII) comprising one ormore OH-protecting groups; or alternatively, (a2) coupling a linkercompound comprising a moiety of structure E with a polyethylene glycolderivative selected from PEG-OH, PEG-NH₂, PEG-CO₂H, and PEG-CO₂R¹⁰ toform an intermediate; and (b2) coupling the intermediate with aglucocorticoid hydrazone derivative of formula (A) or (B) to form aprecursor compound to the compound of structure (XII) comprising one ormore OH-protecting groups; and

(c) removing the OH-protecting groups from the precursor compound toobtain the compound of structure (XII), wherein R¹⁰ is C₁-C₄ alkyl. 23.The method of claim 22, comprising the steps of: (a1) coupling a linkercompound comprising a moiety of the structure E with an OH-protectedglucocorticoid hydrazone derivative of formula (A) or (B) to form anintermediate; and (b1) coupling the intermediate with a polyethyleneglycol derivative selected from PEG-OH, PEG-NH₂, PEG-CO₂H, andPEG-CO₂R¹⁰ to form a precursor compound to the compound of structure(XII) comprising one or more OH-protecting groups.
 24. The method ofclaim 22, comprising the steps of: (a2) coupling a linker compoundcomprising a moiety of the structure E with a polyethylene glycolderivative selected from PEG-OH, PEG-NH₂, PEG-CO₂H, and PEG-CO₂R¹⁰ toform an intermediate; and (b2) coupling the intermediate with anOH-protected glucocorticoid hydrazone derivative of formula (A) or (B)to form a precursor compound to the compound of structure (XII)comprising one or more OH-protecting groups.
 25. The method of claim 22,or a pharmaceutically acceptable salt thereof, wherein n is 40 to 50.26. The method of claim 22, wherein the glucocorticoid hydrazonederivative (A) is selected from:

and wherein the glucocorticoid hydrazone derivative (B) is selectedfrom:

wherein any of PG¹ is an OH-protecting group.
 27. The method of claim22, wherein the compound of structure (XII) is a compound of formula(VIII), (IX), (X), or (XI):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is C₁-C₆alkyl; and R is a molecular moiety of glucocorticoid hydrazonederivative selected from:


28. The method of claim 22, wherein the compound of structure (XII) is acompound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein R¹, A, B, D, E,Z, Y, X, T, Q, P, G, m, and w are as defined in claim
 22. 29. The methodof claim 28, wherein the compound is a compound of formula 7:

or a pharmaceutically acceptable salt thereof.
 30. The method of claim29, comprising the steps of: (a) coupling OH-protected dexamethasonehydrazone derivative 4 with an NH₂-protected glutamic acid S1 to form acompound 5, wherein PG¹ is an OH-protecting group, and PG² is anNH₂-protecting group; (b) removing the protecting group PG² from thecompound 5 to form a compound 6; (c) coupling the compound 6 with apolyethylene glycol-derived carboxylic acid derivative mPEG-CO₂H to forma compound 7a; and (d) removing the protecting group PG¹ from thecompound 7a to obtain the compound 7, wherein the method issubstantially illustrated in the reaction scheme below:

 wherein R is


31. The method of claim 30, wherein the OH-protecting group PG¹ istert-butyldimethylsilyl (TBS), and the NH₂-protecting group PG² isfluorenylmethoxycarbonyl (Fmoc).
 32. The method of claim 31, wherein thedeprotection of step (b) comprises a reaction with piperidine, and thedeprotection of step (d) comprises a reaction with tetra-n-butylammoniumfluoride (TBAF).
 33. The method of claim 28, wherein the compound is acompound of formula 9:

or a pharmaceutically acceptable salt thereof.
 34. The method of claim33, comprising the steps of: (a) coupling a citrate monoester with anOH-protected dexamethasone hydrazone 2 to form a compound 8; (b)coupling the compound 8 with a polyethylene glycol amine derivative(mPEG-NH₂) to form a compound 9a; and (c) removing the OH-protectinggroup from the compound 9a to obtain the compound 9; wherein thereaction steps are substantially illustrated in the reaction schemebelow:

wherein: R is

 and PG¹ is an OH-protecting group.
 35. The method of claim 34, whereinthe OH-protecting group PG¹ is tert-butyldimethylsilyl (TBS), and thedeprotection of step (c) comprises a reaction with tetra-n-butylammoniumfluoride (TBAF).
 36. The method of claim 28, wherein the compound is acompound of formula 11:

or a pharmaceutically acceptable salt thereof.
 37. The method of claim36, comprising the steps of: (a) coupling a pentaerythritol derivativeS3 with a polyethylene glycol derivative mPEG-NH₂ to form a compound 10;(b) hydrolyzing the compound 10 to form a tricarboxylic acid 10a; (c)coupling the compound 10a with an OH-protected dexamethasone hydrazonederivative 2 to form an intermediate; and (d) removing the OH-protectinggroup from the intermediate of step (c) to form the compound 11; whereinthe reaction steps are substantially illustrated in the reaction schemebelow:

wherein: R is C₁-C₄ alkyl; and PG¹ is an OH-protecting group.
 38. Themethod of claim 37, wherein R is methyl; the OH-protecting group PG¹ istert-butyldimethylsilyl (TBS); and the deprotection of step (d)comprises a reaction with tetra-n-butylammonium fluoride (TBAF).
 39. Themethod of claim 28, wherein the compound is a compound of formula 15:

or a pharmaceutically acceptable salt thereof, wherein R′ is


40. The method of claim 39, comprising the steps of: (a) coupling anNH₂-protected glutamic acid S1 with a compound 6 to form a compound 14;(b) removing the NH₂-protecting group PG² from the compound 14 to form acompound 14a; (c) coupling the compound 14a with a polyethylene glycolcarboxylic acid derivative mPEG-CO₂H to form a compound 15a; and (d)removing the OH-protecting group PG¹ from the compound 15a to form thecompound 15; wherein the reaction steps are substantially illustrated inthe reaction scheme below:

wherein: R is

R′ is

PG¹ is an OH-protecting group, and PG² is an NH₂-protecting group. 41.The method of claim 40, wherein the OH-protecting group PG¹ istert-butyldimethylsilyl (TBS), and the NH₂-protecting group PG² isfluorenylmethoxycarbonyl (Fmoc).